PROJECT SUMMARY Functional MRI (fMRI) is performed at a macroscopic scale of 1 to 3 millimeters spatial resolution. The term `mesoscale' has come to denote the resolution of a finer granularity of neuronal organization, to show functional organization across the depth and along the surface of the cortex. Mesoscale fMRI representation of neural activity, however, is not firmly established. A primary objective of this research is to evaluate fMRI's ability to accurately differentiate neuronal activity in cortical layers and columns. This will allow studies of local circuitry in columnar organization and layers with fiber projections to and from distant brain regions, so that hierarchical and directional connectivity between hundreds of human brain regions may eventually be routinely studied non-invasively in the human brain. This project will leverage state-of-the-art MRI hardware and pulse sequences specifically designed for high- resolution imaging of human cortex in a BRAIN Initiative project for next generation human brain imaging (NIH R24MH106096 ?MRI Corticography? (MRCoG)). It will also use several cutting-edge neuroscience technologies, including CLARITY, optogenetic fMRI (ofMRI), transcranial magnetic stimulation (TMS) and electrocorticography (ECoG), to identify and manipulate neuronal activity underlying the fMRI signal. To determine the spatial specificity and laminar profile of BOLD activity, we will use optogenetic stimulation of neuronal populations in different cortical layers of mouse brain while simultaneously imaging with BOLD fMRI. Secondly, variations of vascular and neuronal density will be disambiguated from variations of co-localized fMRI activity using CLARITY and 3D fluorescence microscopy. In humans, the microscale to mesoscale fMRI mapping will be validated using direct electrophysiological mapping with ultra-high-density ECoG grids and advanced computational modeling. To elucidate whole brain mesoscale circuit interactions in humans, MRCoG will be combined with TMS to test hierarchical organization of frontal cortex and transhemispheric motor connections. In humans, pharmacologically modulated brain circuits will be evaluated using an FDA-approved cholinesterase inhibitor, to determine the laminar profile of mesoscale fMRI when feedforward processing is increased.